Plasmodium falciparum gametocyte production correlates with genetic markers of parasite replication but is not influenced by experimental exposure to mosquito biting

Summary Background Plasmodium blood-stage parasites balance asexual multiplication with gametocyte development. Few studies link these dynamics with parasite genetic markers in vivo; even fewer in longitudinally monitored infections. Environmental influences on gametocyte formation, such as mosquito exposure, may influence the parasite's investment in gametocyte production. Methods We investigated gametocyte production and asexual multiplication in two Plasmodium falciparum infected populations; a controlled human malaria infection (CHMI) study and a 28-day observational study in naturally infected individuals in Burkina Faso with controlled mosquito exposure. We measured gene transcript levels previously related to gametocyte formation (ap2-g, surfin1.2, surfin13.1, gexp-2) or inhibition of asexual multiplication (sir2a) and compared transcript levels to ring-stage parasite and mature gametocyte densities. Findings Three of the five markers (ap2-g, surfin1.2, surfin13.1) predicted peak gametocytaemia in the CHMI study. An increase in all five markers in natural infections was associated with an increase in mature gametocytes 14 days later; the effect of sir2a on future gametocytes was strongest (fold change = 1.65, IQR = 1.22–2.24, P = 0.004). Mosquito exposure was not associated with markers of gametocyte formation (ap2-g P = 0.277; sir2a P = 0.499) or carriage of mature gametocytes (P = 0.379). Interpretation All five parasite genetic markers predicted gametocyte formation over a single cycle of gametocyte formation and maturation in vivo; sir2a and ap2-g were most closely associated with gametocyte growth dynamics. We observed no evidence to support the hypothesis that exposure to Anopheles mosquito bites stimulates gametocyte formation. Funding This work was funded by the 10.13039/100000865Bill & Melinda Gates Foundation (INDIE OPP1173572), the European Research Council fellowship (ERC-CoG 864180) and UKRI Medical Research Council (MR/T016272/1) and Wellcome Center (218676/Z/19/Z).

) and with exposure in past 6 months (Rh2.2030,GEXP18, Etramp5.Ag1) were determined at baseline in the Burkina Faso cohort; at day 0 (n = 115) or day 14 (n = 4) of follow-up.A multilevel model was used with an interaction term between antibody response for a specific antigen and ring-stage parasite density on timepoint T to assess the relation to ring-stage parasite density 14 days later (i.e.timepoint T +14 ).Ring-stage parasite density at T +14 was the model outcome variable.Antibody responses against the six antigens were included as categorical variables in separate models.For each antigen separately, the first quartile (lowest 25% in mean fluorescent intensity) was used as reference category and compared to the fourth quartile (upper 25%).For every antigen, the estimate (β) indicates the difference in the relation between current and future ring-stage parasites (on timepoint T +14 ) for the upper 25% compared to the lower 25% with associated P-values.All models accounted for an effect of age (in categories 5-9 years, 10-15 years, 16 years and above).
Mature gametocyte density per μL on T +14 ), a multilevel model was used with combinations of three markers (ap2-g, gexp-2, sir2a) at T as a predictor variable and gametocyte density at T +14 as an outcome variable.The model accounted for concurrent ring-stage parasites (sbp-1) and gametocytes, and included a random person effect to account for multiple observations from the same individual.Fold change indicates the change in outcome variable when the predictor variable would increase 10 fold, 95% confidence interval (CI) indicates the range of fold changes.p-values below 0.05 were considered significant and R 2 indicates the goodness of fit.

Table 3 :
Genetic markers and their previously described function and parasite phase of expression.

Table 4 :
Marker transcripts in vitro.Transcripts of 11 genetic markers were detected using qPCR in percoll gradient synchronized NF54 3D7 P. falciparum culture material.Blood-stage parasites were harvested at 10, 20, 30, and 40 hours to respectively collect early and late-ring stage parasites, mature trophozoites, and schizonts.Material was treated with Nacetylglucosamine to eliminate asexual parasites and obtain mature gametocytes at day 16 post synchronization.Transcript levels were assessed using two concentrations per parasite stage.Per reaction, CT levels are indicated and an N/A is given when zero transcripts were detected.

Table 5 :
Genetic marker levels in a pilot subset of samples from Burkina Faso.To assess marker transcript suitability in field samples, we selected a subset (n=7) of samples from the longitudinal follow-up study in Burkina Faso.This pilot selection included samples negative for both ring-stage parasites (sbp-1) and gametocytes (n=3, sample IDs 1-3), samples that were ring-stage negative yet gametocyte positive (n=2, background signal in StV gametocytes, samples IDs 4 and 5), and samples without gametocytes yet with ring-stage parasites and of which the following sample was gametocyte positive (n=2, 'clean commitment', sample IDs 6 and 7).Values indicate CT-values per reaction, N/A indicates no CT was detected.Reactions with wrong melttemperatures were excluded.

Table 7 :
Demographics of asymptomatic study participants from the longitudinal cohort study in Burkina Faso.Age is reported in age categories and participant sex categories indicate the sex assigned at birth.Individuals aged 5 years or above were eligible for screening.

Table 6 :
Demographics of study participants from controlled human malaria infection (CHMI) study in the Netherlands.Study participants were all aged between 20 and 30 years.Age is reported as the median of total study participants or by sex assigned at birth.

Table 8 :
Ring-stage parasite and gametocyte prevalence at enrolment in the longitudinal cohort study in Burkina Faso, separated by age category.

Table 9 :
Future ring-stage parasites in relation to antibody responses against antigens that reflect cumulative and short-term exposure to Plasmodium falciparum parasites.Antibody responses against antigens associated with cumulative exposure (AMA-1, MSP-1.19,GLURP.R2

Table 10 :
Future gametocyte densities in relation to combinations of marker transcript densities.To assess whether combinations of marker transcripts at timepoint T relate to gametocyte densities 14 days later (T +14